Process for preparing streptokinaserich material



United States Patent 3,419,472 PROCESS FOR PREPARING STREPTOKINASE- RICHMATERIAL Pentti Kasper Siiteri, Dallas, Tex., and Richard Douglas Mills,Pearl River, N.Y., assignors to American Cyanamid Company, Stamford,Conn., a corporation of Maine No Drawing. Original application Oct. 24,1960, Ser. No. 64,655, now Patent No. 3,226,304, dated Dec. 28, 1965.Divided and this application Sept. 20, 1965, Ser. No. 488,752 Theportion of the term of the patent subsequent to Dec. 28, 1982, has beendisclaimed 18 Claims. (Cl. 195-68) This application is a division of ourcopending application Ser. No. 64,655, filed Oct. 24, 1960, now US.Patent 3,226,304, issued Dec. 28, 1965, which in turn is acontinuation-in-part of our copending application Ser. No. 711,867,filed J an. 29, 195 8, now abandoned.

This invention relates to preparing streptokinase-rich material fromrelatively impure sources thereof.

streptokinase, a substance capable of activating plasminogen to plasminwhich has fibrinolytic activity, is produced by many strains ofhemolytic streptococci, chiefly those of Group A and Lancefield Group Cstreptococci.

With a view toward separating streptokinase from other and frequentlyundesirable co-produced metabolic by-products so as to obtain astreptokinase-rich material for potential as yet untried procedures inthe control of thrombo-embolic disease and related disease, numerousprior art efforts have fallen short of providing an especially richproduct. In contrast, the present invention relatively speaking, makespossible a material exceptionally rich in streptokinase.

We have found unexpectedly that a streptokinase material free to aremarkable and heretofore unattained degree, may be obtained by means ofa one-cycle adsorption-elution process employing a cellulose amine anionexchanger and phosphate buifer under carefully controlled conditions.The resulting material is useful in medicinal applications wherestreptokinase type activity acts to enchance fibrinolysis at the site ofan injury.

The process of our invention is based on the general principle ofadsorption chromatography as applied to anion exchange columnadsorbants. The column adsorbants may be prepared from commercialcellulose by the successive steps of treating it with alkali to renderit more reactive and treating the activated cellulose with an aqueoussolution of a reactive amine to provide for ionizable substituent groupsbeing attached to the cellulose moiety by way of ether linkages. Forexample, the reactive amine may be 2-chlorotriethylamine. The modifiedcellulose thus obtained is washed in turn with aqueous alkali solution,mild aqueous acid solution, again with mild aqueous alkali solution andfinally with water. The nitrogen content (Dumas Method) of the resultantwater-washed and dried modified cellulose is of the order of about 1%nitrogen, although any such product assaying between 0.7 and 1.5% may beused satisfactorily. The modified cellulose material may be furthercharacterized by its ion exchange capacity as determined by directtitration against standard hydrochloric acid solution. For instance, anormal product of this type will titrate about 0.71 milliequivalent pergram of the modified cellulose. The modified cellulose may be used incolumns of from 1 cm. and greater in diameter with the height of thecolumn bed being approximately ten to fifteen times its diameter. Beforethe modified cellulose is put in the column, however, it is conditionedby slurrying in water and adjusting the pH thereof to an arbitrary valuewith an aqueous acidic reagent. It is then further conditioned bywashing with buffer solution of the same arbitrary pH. The bufferedcellulose slurry is poured into the column, allowed to settle by gravityand finally packed with the aid of compressed air. The column is thenready for use.

A solution of crude streptokinase material that has been 'dialyzedagainst a portion of the identical buffer solution used in theconditioning of the modified cellulose is now placed on top of thecolumn bed and allowed to flow into the column by gravity. After thestreptokinase solution has completely flowed into the column bed anarbitrary amount of the same buffer solution is placed on top of thecolumn bed to provide a head of fluid above the column bed.Simultaneously, the desorption of the adsorbed streptokinase activity isbegun by means of standard elution technique whereby a buffer isintroduced at the top of the column and the efliuent from the column iscollected in successive fractions. As this buffer flows through thecolumn, the streptokinase activity moves down the column and finally isrecovered from the bottom of the column in the form of a dilute aqueoussolution.

The type of butter used to purify streptokinase by the above-describedprocess is very critical. We have found that only the phosphate type issuitable for our purpose.

The molarity of the phosphate buffer used to condition the column bedprior to commencement of elution is also critical, the permissiblemaximum value being 0.1 M. Molarity greater than 0.1 will prevent properadsorption of streptokinase activity by the modified cellulose bed andthereby result in improper separation of streptokinase from impuritiesassociated therewith. Below 0.005 M the streptokinase activity is notdesorbed properly.

The pH of the phosphate butter solution used to condition the modifiedcellulose column should be kept between about 5.8 and 8.5. Above aboutpH 8.5 the ionization characteristics of the modified cellulose aregradually suppressed and, consequently, the capacity of the cellulose toadsorb streptokinase is concomittantly suppressed. Below about pH 5.8streptokinase itself tends to precipitate inasmuch as its isoelectricpoint is in that range.

The temperature range within which the elution technique described abovemay be applied successfully to crude streptokinase material is 0 C. to10 C. The preferred range is 3 C. to 5 C. Above 10 C. denaturation ofstreptokinase appears to take place.

While, as stated above, the standard elution technique may be used, in apreferred embodiment of the process of our invention gradient elution isemployed. As is well known, in gradient elution a buffer of graduallychanging pH and/or molar strength is introduced into the column forpurposes of elution.

The extent to which the molarity of the phosphate buffer solution mayvary during the gradient elution process is considerable, a top value ofabout 0.4 being considered a practical upper limit. It must vary to someextent, however, in order for the process to be successful. The pH mayvary from a preferred starting value of between 6.0 to 6.5 to about 8.5on the high side and 5.8 on the low side, for the reasons outlined aboveconcerning the pH limitations of the buffer solution used to conditionthe column at the outset. The flow rate through the column is notcritical but a value recommended is 3 ml./ sq. cm. of cross-sectionalarea/hour.

Regarding the starting material useful for the process of our invention,it is to be understood that the term crude streptokinase is meantstreptokinase-bearing material such as ordinary commercially availablestreptokinase-streptodornase mixtures as well as the up-gradedstreptokinase material prepared by the process claimed in U.S. Patent2,784,145 to Ablondi and Mills. Numerous other partially purifiedstreptokinase preparations, such as those that would occur to oneskilled in the art, would likewise be suitable starting materials forour process.

The following examples will serve to illustrate the scope of ourinvention with more particularity.

EXAMPLE 1 137 grams of crude streptokinase assaying about 80streptokinase units per gamma of nitrogen were suspended in water andbrought into solution by the addition of dilute sodium hydroxide. Thissolution was dialyzed against 0.05 sodium phosphate at pH 6.5. Thesolution was removed from the dialysis bag and a small amount ofinsoluble material was removed by centrifugation. The volume of thissolution was 485 ml. and it contained 30-5 million streptokinase units.The dialyzed solution was divided into two equal portions and eachportion was allowed to run by gravity into two identical columnsprepared with a cellulose anion exchanger which had been made byreacting cellulose with Z-chlorotriethylamine. These columns had aninternal diameter of 3.7 ems. and the height of the column bed wasapproximately 40 cms. Gradient elution with increasing pH and molarstrength was used to remove the streptokinase activity from the columnsby the constant column technique. Both mixing bottle and reservoir ofthe gradient device were of similar shape and cross-sectional area. Themixing bottle contained 1800 mls. of 0.05 M phosphate buffer at pH 6.5and the reservoir contained 1800 mls. of solution which was 0.1 M withrespect to Na HPO and 0.2 M with respect to NaCl. The flow rate throughthe columns was approximately 3 ml./sq. cm. per hour. Thestreptokinase-rich fraction from each column contained approximately 145million streptokinase units. Two additional columns were run in asimilar manner starting with 180 grams of crude streptokinase. Thestreptokinase-rich fractions from these four columns were combined andthe streptokinase was precipitated therefrom by the addition of sodiumchloride and adjustment of the pH of the solution to a value of 4.0.

EXAM PLE 2 34 grams of crude streptokinase assaying 100 streptokinaseunits per gamma of nitrogen were purified by batch-wise adsorption andelution at pH 7.0 from a modified cellulose anion exchanger, theexchanger as fully described in Example 1. Further purification of thispartially purified streptokinase was achieved by gradient elutionchromatography on the same type of modified cellulose as follows: Asolution of the partially purified streptokinase was dialyzed against0.075 M sodium phosphate at pH 6.0 and then allowed to flow into acolumn of cellulose anion exchanger. The inside diameter of this columnwas 3.7 cm. and the height of the column was 55 cm. Gradient elution ofthe streptokinase was achieved by a system in which the mixing bottlecontained 2000 ml. of 0.075 M sodium phosphate at pH 6.0 and thereservoir contained 200 ml. of a solution that was 0.2 M with respect toboth NaH PO and NaCl.

EXAMPLE 3 25 grams of crude streptokinase assaying about 150streptokinase units per gamma of nitrogen were suspended in water anddissolved by the addition of dilute sodium hydroxide. This solution wasdialyzed against 0.04 M phosphate buffer, pH 7.0, until the pH insidethe dialyzing bag was 7.0 and the phosphate buffer concentration was0.04 M. The volume of this solution was 70 ml. and it contained 122.6million units of streptokinase. The dialyzed solution was allowed to runby gravity into a column of modified cellulose anion exchanger. Thecolumn had an internal diameter of 3.7 cms. and the height of the columnwas 46.0 cms. Elution of impurities was achieved by passing about 800ml. of 0.075 M phosphate buffer, pH 8.0, through the column, under whichconditions the streptokinase is not yet desorbed. Flow rate was asdescribed in previous examples. When the impurities were eluted theelution with 0.075 M phosphate buifer was discontinued and elution ofthe streptokinase begun by flowing 0.1 M phosphate buffer, pH 7.0,through the column. About 800 ml. of this buffer were used for elution.The streptokinase-rich fraction containing 93 million units wascollected.

We claim:

1. A one-cycle adsorption-elution process for preparingstreptokinase-rich material from relatively impure sources thereof whichconsists in the steps of (a) adsorbing crude streptokinase on acellulose amine anion exchanger and (b) desorbing purified streptokinasetherefrom by means of elution, said preceding steps taking place in thepresence of a phosphate buffer system having a pH value of not less thanabout 5.8 and not more than about 8.5, a temperature of not less than 0C. and not more than 10 C., and a molarity of not less than about 0.005and during said adsorbing step of not more than about 0.1 and duringsaid desorbing step of not more than about 0.4, and recovering thestreptokinase-rich material from the eluate.

2. The process of claim 1 wherein said elution is a gradient elution.

3. The process of claim 1 wherein said elution is a twostage elutionconducted firstly with said system adjusted to about 0.075 M phosphateand about pH 8 to remove impurities and secondly with said systemadjusted to about 0.1 M phosphate and about pH 7 to removestreptokinase-rich material. v

4. The process of claim 2 wherein said cellulose amine anion exchangeris used in the form of a column.

5. The process of claim 3 wherein said cellulose amine anion exchangeris used in the form of a column.

6. The process of claim 4 wherein said temperature is not less than 3 C.and not more than 5 C.

7. The process of claim 5 wherein said temperature is not less than 3 C.and not more than 5 C.

8. The process of claim 4 wherein the cellulose amine anion exchangercomprises the reaction product of activated cellulose with2-chlorotriethylamine.

9. The process of claim 5 wherein the cellulose amine anion exchangercomprises the reaction product of activated cellulose with2-chlorotriethylamine.

10. A one-cycle adsorption-elution process for purifying streptokinasewhich consists essentially in (at) bringing into contact with acellulose amine anion exchanger a solution of streptokinase in aphosphate buffer having a molar concentration of from 0.005 to 0.1 and(b) contacting the cellulose amine anion exchanger with a phosphatebuffer having a molar concentration between 0.005 and 0.4 to selectivelyremove streptokinase, said phosphate buffer having a pH value of notless than about 5 .8 and not more than about 8.5 and a temperature ofnot less than 0 C. and not more than 10 C.

11. The process of claim 10 wherein the cellulose amine anion exchangeris used in the form of a column.

12. The process of claim 10 wherein the temperature is not less than 3C. and not more than 5 C.

13. The process of claim 10 wherein the cellulose amine anion exchangercomprises the reaction product of activated cellulose and2-chlorotriethylamine.

14. The process of claim 10 wherein the selective removal ofstreptokinase includes the use of gradient elution technique.

15. A one-cycle adsorption-elution process for purifying streptokinasewhich consists essentially in (a) bringing into contact with a celluloseamine anion exchanger a solution of streptokinase in a phosphate 5 6bulfer having a molar concentration of from 0.005 18. The process ofclaim 15 wherein the cellulose to 0.1, amine anion exchanger comprisesthe reaction product of (b) washing the cellulose amine anion exchangerwith activated cellulose and 2-chlorotriethylamine.

a phosphate buffer having a molar concentration of approximately 0.075to remove impurities, and 5 References Cited (c) removing streptokinasefrom the cellulose amine UNITED STATES PATENTS anion exchanger with aphosphate buffer having a molar concentration of about 0.1, 3,226,30412/ 1965 Siiteri et al 195 said phosphate buffer having a pH value ofnot less than about 5 .8 and not more than about 8.5 and a temperature10 A RT T- MYERS, i ary Examiner. of not less than 0 C. and not morethan 10 C.

16. The process of claim 15 wherein the cellulose amine FRIEDMANAs'slstant Exammer' anion exchanger is used in the form of a column. U SQ X R 17. The process of claim 15 wherein the temperature is not lessthan 3 C. and not more than 5 C, 5 424- 94

1. A ONE-CYCLE ABSORPTION-ELUTION PROCESS FOR PREPARINGSTREPTOKINASE-RICH MATERIAL FROM RELATIVELY IMPURE SOURCES THEREOF WHICHCONSISTS IN THE STEPS OF (A) ADSORBING CRUDE STREPTOKINASE ONA CELLULOSEAMINE ANION EXCHANGE AND (B) DESORBING PURIFIED STREPTOKINASE THEREFROMBY MEANS OF ELUTION, SAID PROCEEDING STEPS TAKING PLACE IN THE PRESENCEOF A SAID PRECEEDING BUFFER SYSTEM HAVING A PH VALUE OF NOT LESS THANABOUT 5.8 AND NOT MORE THAN ABOUT 8.5, A TEMPERATURE OF NOT LESS THAN0*C. AND NOT MORE THAN ABOUT 10*C., AND A MOLARITY OF NOT LESS THANABOUT 0.1 AND DURING SAID DESORBING STEP OF NOT MORE THAN ABOUT 0.4, ANDRECOVERING THE STREPTOKINASE-RICH MATERIAL FROM THE ELUATE.